A variety of methods for nucleic acid amplification have been developed as techniques that allow detection of very small amounts of nucleic acids in a sample. In particular, a method in which efficient isothermal amplification is carried out (e.g., the LAMP method) has been employed in a wide variety of testing fields as a technique that allows simple and rapid detection with the use of a cost-effective apparatus without a temperature control function.
The present inventors reported that nucleic acids could be quantified at a concentration range of 103 to 109 copies/test with the use of a LAMP detection system that does not involve the use of an internal standard material (Y. Mori, et al., Journal of Biochemical and Biophysical Methods, 59, 145-157, 2004). Since this technique does not use an internal standard material, the results obtained by such technique are relative amounts of target nucleic acids.
Meanwhile, a method of quantification involving the use of an internal standard material in the LAMP method was reported (H. Tani, et al., Analytical Chemistry, 79 (15), 5608-5613, 2007). This method involves the use of an internal standard material having a sequence similar to that of the target nucleic acid to simultaneously amplify the target nucleic acid and the internal standard material. Since the quantifiable concentration range of this method is as small as 104 to 108 copies/test, it is necessary to dilute a sample and repeat assays a plurality of times in order to assay nucleic acids of a wide concentration range.